Host cell lines expressing human recombinant growth and differentiation factor-5 (rhGDF-5)

ABSTRACT

Expression vector systems are provided for increased production of a recombinant GDF-5 (rhGDF-5) protein. Also provided are transformed host cells that were engineered to produce and express high levels of rhGDF-5 protein. Methods for isolating recombinant GDF-5 protein from an inclusion body of a cell are disclosed herein. The methods as disclosed are cost-effective, time-saving and are of manufacturing quality.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 13/750,793, filed Jan. 25, 2013, now U.S. Pat. No. 9,169,308entitled “METHODS AND COMPOSITIONS OF HUMAN RECOMBINANT GROWTH ANDDIFFERENTIATON FACTOR-5 (RHGDF-5) ISOLATED FROM INCLUSION BODIES,” whichis incorporated herein by reference in its entirety.

BACKGROUND

The present disclosure relates generally to a recombinant human growthand differentiation factor-5 (rhGDF-5) protein and, specifically toexpression vector systems for increased production of rhGDF-5, hostcells or cell lines for producing rhGDF-5, methods of producing rhGDF-5using the host cells or cell lines and methods of enhancing productionand protein expression of rhGDF-5 protein that are cost-effective,time-saving and manufacturing quality.

Biologic, a therapeutic product, can be made by genetically engineeringliving cells and requires a high level of precision and care and variousfactors for its manufacturing process to yield a consistent biologicproduct each time. For example, a biologic that is produced byrecombinant host cells, either in prokaryotes or eukaryotes, can beinfluenced by (i) individual cell characteristics and (ii) theenvironment and nutrients provided during the manufacturing process. Anexample of a biologic is Growth and Differentiation Factor-5 (GDF-5).

GDF-5 belongs to the Bone Morphogenetic Protein (BMP) family, whichitself is a subclass of the transforming growth factor-β superfamily ofproteins. There are several variants and mutants of GDF-5 (GDF familymembers), some of which include the first isolated mouse GDF-5 (U.S.Pat. No. 5,801,014); MP52, a human form of GDF-5 (hGDF-5; (WO 95/04819))or LAP-4 (Triantfilou et al., Nature Immunology 2, 338-345, 2001);cartilage-derived morphogenetic protein (CDMP)-1, an allelic proteinvariant of hGDF-5 (Chang, S. C. et al., J. Biol. Chem. 269(45):28227-34(1994); WO 96/14335); rhGDF-5, a recombinant human form prepared frombacteria (EP 0955313); rhGDF-5-Ala83, a monomeric variant of rhGDF-5;BMP-14, a collective term for hGDF-5/CDMP-1 like proteins; SYNS2;Radotermin, the international non-proprietary name designated by theWorld Health Organization; HMW MP52's, high molecular weight proteinvariants of MP52; C465A, a monomeric version wherein the cysteineresidue responsible for the intermolecular cross-link is substitutedwith alanine; also other active monomers and single amino acidsubstitution mutants including N445T, L441 P, R438L, and R438K.

The GDF-5 family members share common structural features including acarboxy terminal active domain and is characterized by a polybasicproteolytic processing site, which can be cleaved to release a matureprotein containing seven conserved cysteine residues. The conservedpattern of cysteine residues creates 3 intra-molecular disulfide bondsand one inter-molecular disulfide bond. The active form can be either adisulfide-bonded homodimer of a single family member or a heterodimer oftwo different members (Massague et al., Ann. Rev. Cell Biol. 6:957(1990); Sampath et al., J. Biol. Chem. 265:13198 (1990); Celeste et al.,Proc. Natl. Acad. Sci. USA 87:9843-7 (1990); U.S. Pat. No. 5,011,691 andU.S. Pat. No. 5,266,683). The proper folding of the GDF-5 protein andformation of these disulfide bonds are essential to biologicalfunctioning, and misfolding leads to inactive aggregates and cleavedfragments.

GDF-5 is expressed in the developing central nervous system (O'Keeffe,G. et al., J. Neurocytol. 33(5):479-88 (2004) and has a role in skeletaland joint development (Buxton, P. et al., J. Bone Joint Surg. Am. 83-A,S1(Pt. 1):523-30 (2001); Francis-West, P. et al., Development126(6):1305-15 (1999); Francis-West, P. et al., Cell Tissue Res.296(1):111-9 (1999)). The GDF-5 family members are regulators of cellgrowth and differentiation in both embryonic and adult tissues. Forexample, GDF-5 may induce angiogenesis in the bone formation process(Yamashita, H. et al., Exp. Cell Res. 235(1):218-226 (1997); CDMP-1stimulates activity of articular chondrocytes thereby contributing tothe integrity of the joint surface (Erlacher, L. et al., ArthritisRheum. 41(2):263-73 (1998)). Changes in expression patterns of GDF-5 andits receptors are associated with human articular chondrocytededifferentiation (Schlegel, W. et al., J. Cell Mol. Med.13(9B):3398-404 (2009)). As a growth factor, GDF-5 (CDMP) may stimulateproteoglycan production in the human degenerate intervertebral disc (LeMaitre, C. L. et al., Arthritis Res. Ther. 11(5):R137 (2009)). It mayincrease the survival of neurons that respond to a dopamineneurotransmitter and can be a potential therapeutic molecule associatedwith Parkinson's disease. (Sullivan and O'Keeffe, J. Anat. 207(3):219-26(2005)). When rhGDF-5 was delivered on beta-tricalcium phosphate, aneffective encouragement of periodontal tissue regeneration in non-humanprimates was observed. In tissues critical for periodontal repair (e.g.alveolar bone, cementum and periodontal ligament), rhGDF-5 treatment onthese tissues showed evidence of regeneration and the response was foundto be dose-dependent (Emerton, K. B. et al., J. Dental Res.90(12):1416-21 (2011). Based on this finding and other similar reports,a biologic such as GDF-5 may offer new approaches or options toregenerate bone during dental implant placement and may save a tooth inpatients who are at risk for tooth loss due to periodontal disease.

GDF-5 gene mutations can be associated with the following healthconditions, e.g., acromesomelic chrondrodysplasia Grebe type (AMDG;(Thomas, J. T. et al., Nat. Genet. 1:58-64 (1997);), Hunter-Thompsontype (AMDH; (Thomas, J. T. et al., Nat. Genet. 3:315-7 (1996));brachydactyly type C (BDC; Francis-West, P. H. et al., Development,126(6):1305-15 (1999), Everman, D. B. et al., Am. J. Med. Genet.,112(3):291-6 (2002), Schwabe, G. C. et al., Am. J. Med. Genet. A.124A(4):356-63 (2004)); DuPan syndrome (DPS), which is also known asfibular hypoplasia and complex brachydactyly (Faiyaz-Ul-Haque, M. etal., Clin. Genet. 61(6):454-8 (2002)); Mohr-Wriedt brachydactyly type A2(Kjaer, K. W. et al., J. Med. Genet. 43(3):225-31 (2006)); multiplesynostoses syndrome type 2 (SYNS2; Dawson, K. et al., Am. J. HumanGenet. 78(4):708-12 (2006), Schwaerzer, G. K. et al., J. Bone Miner.Res. 27(2):429-42 (2012)); semidominant brachydactyly A1 (BA1; Byrnes,A. M. et al., Hum. Mutat. 31(10):1155-62 (2010)); symphalangism (SYM1;Yang, W. et al., J. Hum. Genet. 53(4):368-74 (2008)) or brachydactylytype A2 (BDA2; Seemann, P. et al., J. Clin. Invest. 115(9):2373-81(2005), Plöger, F. et al., Hum. Mol. Genet. 53(4):368-74 (2008));susceptibility to osteoarthritis type 5 (OS5; Masuya, H. et al., Hum.Molec. Genet. 16:2366-75 (2007), Miyamoto, Y. et al., Nature Genet.39:529-53 (2007)); knee osteoarthritis in Thai ethnic population(Tawonsawatruk, T. et al., J. Orthop. Surg. Res. 6:47 (2011)). GDF5 genevariants have been associated with hand, knee osteoarthritis andfracture risk in elderly women, which replicates the previousassociation between GDF5 variation and height. (Vaes, R. B. et al., AnnRheum. Dis. 68(11):1754-60 (2009)). All of these associations confirmedthat the GDF-5 gene product may play a role in skeletal development.

Expression of GDF-5-related proteins using recombinant DNA techniqueshas been done and their purification and production for industrial scalehave also been explored. See for example, Hötten, U.S. Pat. No.6,764,994; Makishima, U.S. Pat. No. 7,235,527; Ehringer, U.S. Pat. No.8,187,837). Both Hötten and Makishima described (1) a complete DNAnucleotide sequence that codes for the TGF-β protein MP-52 and thecomplete amino acid sequence of MP52; and (2) a composition containing apharmaceutically active amount of the MP-52 for wound healing and tissueregeneration, treating cartilage and bone diseases and dental implants.According to Makashina, isolation of pure MP-52 at least with the matureregion from the mixture was difficult (Makashina, column 1, lines59-61). To overcome this obstacle, Makashina constructed a DNA plasmidwherein a codon encoding methionine was linked to the DNA sequence thatencodes for a 119-amino acid residue protein (MP-52) and wherein theN-terminal alanine of the mature MP-52 protein (120-amino acid residue)was eliminated. Ehringer, on the other hand, described an advancedmethod for the efficient prokaryotic production and purification ofGDF-5 related proteins that resulted in better protein yield, highproduct purity and improved industrial applicability. Problemsencountered during the purification and refolding of the GDF-5-relatedproteins in large scale were disclosed and addressed.

The use of prokaryotic expression vectors such as bacterial plasmids forexpressing preventive or therapeutic peptides (biologics) is verycritical and beneficial not only for biochemical research andbiotechnology but even more so for medical therapy. Such use is thebasis of many biologics manufacturing processes. High-cell density (HCD)fermentation methods that employ these processes offer many advantagesover traditional methods in that the final product concentrations arehigher, downtime and water usage are reduced, and overall productivityis improved resulting in lower set-up and operating costs.

The recombinant protein and plasmid DNA production typically involves:(1) bacterial propagation and fermentation production, wherein a plasmidencoding a gene of interest is transformed into a bacterial cell,typically Escherichia coli (E. coli), propagated to make master andworking cell banks, and further grown in a bioreactor (e.g., fermentor)to make production cells that contain high yields of the plasmid; and(2) purification and formulation stability, wherein the production cellsare lysed and plasmid DNA carrying the gene of interest is purified by aplurality of purification methods and formulated for delivery.Expression is particularly higher if the gene of interest is codonoptimized to match that of the target organism, which leads to improvedgene function and increased protein expression, which ultimately leadsto cost-effectiveness of mass producing the recombinant protein.

Plasmid fermentation processes for plasmid production should beoptimized to retain a high percentage of supercoiled plasmid. Otherplasmid forms are difficult to eliminate during purification and theirpresence are undesirable. Fermentation media and processes needs to beoptimized for plasmid yield, plasmid quality and compatibility of theresultant cells for harvest and lysis. There are about threefermentation processes that can be utilized to initiate production,namely: batch, batch-fed or continuous fermentation processes. For alarge scale production, a batch fermentation that generally yields about10-20 mg/L of plasmid DNA has its limitations such as uncontrolledgrowth rates and waste product accumulation (e.g., production of reducedcarbon metabolites such as acetates, lactates and formates) thatultimately would lead to inhibition of bacterial growth. To preventthese issues from occurring and to increase plasmid yield, fed-batch orcontinuous high cell density fermentation can be a better option.Continuous fermentation processes are more conducive to the productionof large amounts of a single product but sterility remains an issue.Fed-batch fermentation begins with a short batch fermentation and isproceeded by the addition of media at a defined rate. It is moreflexible and consistent than the batch method and allows for simpleoptimization of fermentation profiles for each plasmid DNA product. Whenemploying a defined growth rate strategy as a form of feed strategy, afeed media is added at rates that are determined based on apre-established growth profile, wherein the feed is triggered by aninitial DO2 spike (caused by the exhaustion of initial bolus of glucosein the media). Peterson, M. and Brune, M., in BioPharm InternationalSupplements entitled: “Maximizing Yields of Plasmid DNA Processes,” Jun.2, 2008.

Chemically-defined (minimal) media contain known quantities ofingredients added to purified water. The absence of animal-derivedcomponents in chemically-defined media may be more desirable from aregulatory standpoint due to concerns over BSE/TSE (spongiformencephalopathy/transmissible spongiform encephalopathy). They havereproducibility (their components have known chemical structures thatcan allow consistent performance of cells in the medium), greatersimplicity of both downstream processing and the analysis of product andgreater control of feeding strategy when carbon sources are known.

Complex media, on the other hand, are digests of food and agricultureby-products (i.e. protein hydrolysate and yeast extract). They canprovide a majority of needed nutrients to host cell (e.g., Escherichiacoli) fermentation. They may produce high yields at lower costs (thus,more cost-effective) and less control over individual components andpossibly vary from lot-to-lot.

Semi-defined media contain small concentrations of complex ingredientsusually from about 0.05 to about 0.5% added to a chemically definedmedia. Semi-defined media can maximize performance while minimizingdownstream processing issues. Small amount of complex material mayprovide enough nutrients to enhance growth of microorganisms withoutinterfering with recovery or analysis.

Given the role of GDF-5 in cell growth and differentiation, inparticular, skeletal and joint development and bone regeneration, thereis a critical need for a therapeutic rhGDF-5 biologic that can bemanufactured in large scale processes. There is an urgent need forimproving the manufacturing process of rhGDF-5 that can becost-effective, time-saving and manufacturing quality.

SUMMARY

The present disclosure includes methods and compositions for theproduction of rhGDF-5 using the T5 or Trc promoter in the production ofrhGDF-5 for therapeutic applications. The rh-GDF-5 can be easilyproduced in large scale quantities in a cost-effective and time-savingmanner.

In various embodiments, there is a method provided for isolatingrecombinant GDF-5 protein from an inclusion body of a cell. The methodcomprises disrupting the cell via lysis or homogenization such that apGDF-5 or a pGDF-5-Trc-transformed cell releases the inclusion body ofrhGDF-5 protein and recovering the GDF-5 protein from the inclusion bodycomprising an expression vector having a T5 or a Trc promoteroperatively linked to a polynucleotide sequence that encodes a GDF-5protein by contacting the GDF-5 protein with a substrate.

In various embodiments, there is a method provided for isolatingrecombinant GDF-5 protein from an inclusion body of a prokaryotic cell.The method comprises disrupting the prokaryotic cell via lysis orhomogenization such that a pGDF-5 or a pGDF-5-Trc-transformed cellreleases the inclusion body of rhGDF-5 protein and recovering the GDF-5protein from the inclusion body comprising an expression vector having aT5 or a Trc promoter operatively linked to a polynucleotide sequencethat encodes a GDF-5 protein by contacting the GDF-5 protein with asubstrate.

In various embodiments, a host cell line is provided. The cell linecomprises an inclusion body comprising an isolated GDF-5 protein,wherein the host cell includes an expression vector which comprises apolynucleotide encoding for an rhGDF-5 protein under the control of a T5or Trc promoter.

Additional features and advantages of various embodiments will be setforth in part in the description that follows, and in part will beapparent from the description, or may be learned by practice of variousembodiments. The objectives and other advantages of various embodimentswill be realized and attained by means of the elements and combinationsparticularly pointed out in the description and appended claims.

BRIEF DESCRIPTION OF THE FIGURES

In part, other aspects, features, benefits and advantages of theembodiments will be apparent with regard to the following description,appended claims and accompanying drawings where:

FIGS. 1A and 1B are plasmid maps of pGDF5-T5 and pGDF5-Trc expressionvectors, respectively.

FIG. 1C shows a protein alignment of the theoretical amino acid sequenceencoded by pGDF5-Trc and that of a commercially-known rhGDF-5 protein,as set forth in the Sequence Listing as SEQ ID NOs: 4 and 5,respectively.

FIGS. 2A and 2B are agarose gels showing NdeI-linearized plasmid DNAsprepared from pGDF5-T5- and pGDF5-Trc-transformed DH10β and STBL2clones.

FIGS. 3A and 3B are Western blots showing the absence and presence ofGDF-5 protein in the supernatant and pellet fractions of selectedpGDF5-Trc-transformed clones, respectively.

FIG. 3C shows the lanes and samples that correspond to the Western blotsof FIGS. 3A and 3B.

FIG. 4A is a Western blot showing over-expression of GDF-5 inpGDF5-Trc-transformed Clones 1 and 4.

FIG. 4B shows the growth profile and rate of pGDF5-Trc-transformedHMS174 Clones 1 and 4.

FIGS. 5A and 5B shows the growth profiles of pGDF5-Trc-transformedHMS174 Clones 1 and 4 when grown using the ultra yield shake flask and 5L Applikon Fermentor, respectively.

FIG. 6A is a Coomassie brilliant blue-stained gel showing GDF-5 proteinproduction from supernatant and pellet fractions ofpGDF5-Trc-transformed HMS174 Clones 1 and 4 that were grown by eitherusing the ultra yield shake flask method or the 5 L Applikon Fermentormethod.

FIG. 6B is a Western blot showing GDF-5 over-expression from supernatantand pellet fractions of pGDF5-T5- and -Trc-transformed HMS174 Clones 1and 4 that were grown by either using the ultra yield shake flask methodor the 5 L Applikon Fermentor method.

FIG. 7 is an exemplary formulation of a high cell density mediaaccording to the embodiment of the present disclosure.

FIGS. 8A, 8B and 8C show the enhancing effects of sodium molybdate,magnesium sulfate, heptahydrate and sodium chloride on rhGDF-5expression when these three components were added to Media 1, which wasoptimized based on its improved response to rhGDF-5 expression in thecultured pGDF5-Trc-transformed host cells. Data obtained were evaluatedusing statistical software.

FIGS. 9A and 9B show an increase of rhGDF-5 expression by the additionof yeast extract and magnesium sulfate into Media 2 (optimized based onits improved response to rhGDF-5 expression) but the addition of sodiummolybdate decreased rhGDF-5 expression in pGDF5-Trc-transformed hostcells. Data obtained were evaluated using statistical software.

FIGS. 9C and 9D show biomass optimization by the addition of yeastextract while negatively affected by the addition of sodium chloride andMOPS when these three components were added to Media 3, which wasoptimized based on its improved response to biomass yield in the growthof pGDF5-Trc-transformed host cells. Data obtained were evaluated usingstatistical software.

FIGS. 9E and 9F show the results for the optimization of the culturemedia.

FIG. 10 is a Coomassie brilliant blue-stained gel showing the level ofGDF-5 protein production of pGDF5Trc-transformed HMS174 cells when grownunder different types high cell density media that were designed withrespect to their optimized response to either rhGDF-5 expression(protein production) and biomass yield (growth rate): Media 1 (definedmedia improved rhGDF-5 expression); Media 2 (semi-defined media improvedrhGDF-5 expression); and Media 3 (semi-defined media improved biomassyield).

FIGS. 11A-C are Coomassie brilliant blue-stained gels showing the levelof GDF-5 protein production and expression of pGDF5Trc-transformedHMS174 cells when grown under (a) pH 6.5 (condition A); (b) pH 7.1(condition B); and (c) pH 6.8 at low oxygen (condition C), respectively.

FIG. 11D shows the effect of pH and oxygen on protein expression andgrowth rate of pGDF5Trc-transformed HMS174 cells.

FIG. 11E shows the effect of pH and oxygen on the ratio of two majorexpressed bands, 40 kDa:14 kDa of pGDF5Trc-transformed HMS174 cells.

It is to be understood that the figures are not drawn to scale. Further,the relation between objects in a figure may not be to scale, and may infact have a reverse relationship as to size. The figures are intended tobring understanding and clarity to the structure of each object shown,and thus, some features may be exaggerated in order to illustrate aspecific feature of a structure.

DETAILED DESCRIPTION

For the purposes of this specification and appended claims, unlessotherwise indicated, all numbers expressing quantities of ingredients,percentages or proportions of materials, reaction conditions, and othernumerical values used in the specification and claims, are to beunderstood as being modified in all instances by the term “about.”Accordingly, unless indicated to the contrary, the numerical parametersset forth in the following specification and attached claims areapproximations that may vary depending upon the desired propertiessought to be obtained by the present application. At the very least, andnot as an attempt to limit the application of the doctrine ofequivalents to the scope of the claims, each numerical parameter shouldat least be construed in light of the number of reported significantdigits and by applying ordinary rounding techniques.

Notwithstanding that the numerical ranges and parameters setting forththe broad scope of the present disclosure are approximations, thenumerical are as precise as possible. Any numerical value, however,inherently contains certain errors necessarily resulting from thestandard deviation found in their respective testing measurements.Moreover, all ranges disclosed herein are to be understood to encompassany and all subranges subsumed therein. For example, a range of “1 to10” includes any and all subranges between (and including) the minimumvalue of 1 and the maximum value of 10, that is, any and all subrangeshaving a minimum value of equal to or greater than 1 and a maximum valueof equal to or less than 10, e.g., 5.5 to 10.

Additionally, unless defined otherwise or apparent from context, alltechnical and scientific terms used herein have the same meanings ascommonly understood by one of ordinary skill in the art to which thisdisclosure belongs.

Unless explicitly stated or apparent from context, the following termsare phrases have the definitions provided below:

It is noted that, as used in this specification and the appended claims,the singular forms “a,” “an,” and “the” include plural referents unlessexpressly and unequivocally limited to one referent.

For the purposes of this application the term “GDF-5” is meant toinclude all variants and mutants of the GDF-5 protein, and rhGDF-5 is anexemplary member having 125 amino acids as set forth in the SequenceListing as SEQ ID NO:4.

The term “cysteine-knot domain” refers to a conserved cysteine-richamino acid region that is present in the mature parts of TGF-βsuperfamily proteins, such as i.e. human GDF-5 and forms athree-dimensional protein structure known as cysteine-knot. It has beenshown that the cysteine-knot domain alone is sufficient for thebiological function of the protein (Schreuder et al., Biochem. Biophys.Res. Commun. 329:1076-86(2005)). Consensus sequences for cysteine-knotdomains are well known in the state of the art. The cysteine-knot-domainof a protein starts with the first cysteine residue participating in thecysteine-knot of the respective protein and ends with the residue whichfollows the last cysteine participating in the cysteine-knot of therespective protein. For example, the cysteine-knot domain of the humanGDF-5 precursor protein consists of the amino acids 24-125 (see theunderlined region of the amino acid sequence of SEQ ID NO:4 encoded bypGDF5-Trc as shown in FIG. 1C).

The term “recombinant” indicates that the material (e.g., a nucleic acidor a polypeptide) has been artificially or synthetically (i.e.,non-naturally) altered by human intervention. The alteration can beperformed on the material within, or removed from, its naturalenvironment or state. For example, a “recombinant nucleic acid” is onethat is made by recombining nucleic acids, e.g., during cloning, DNAshuffling or other well-known molecular biological procedures. A“recombinant DNA molecule” is comprised of segments of DNA joinedtogether by means of such molecular biological techniques. The term“recombinant protein” or “recombinant polypeptide” as used herein refersto a protein molecule which is expressed using a recombinant DNAmolecule. A “recombinant host cell” is a cell that contains and/orexpresses a recombinant nucleic acid.

A “polynucleotide sequence” or “nucleotide sequence” or “nucleic acidsequence,” as used interchangeably herein, is a polymer of nucleotides,including an oligonucleotide, a DNA, and an RNA, a nucleic acid or acharacter string representing a nucleotide polymer, depending oncontext. From any specified polynucleotide sequence, either the givennucleic acid or the complementary polynucleotide sequence can bedetermined. Included is DNA or RNA of genomic or synthetic origin whichmay be single- or double-stranded and represent the sense or anti-sensestrand.

The term “oligonucleotide” as used herein includes naturally occurring,and modified nucleotides linked together by naturally occurring andnon-naturally occurring oligonucleotide linkages. Oligonucleotides are apolynucleotide subset generally comprising a length of 200 bases orfewer. Preferably oligonucleotides are 10 to 60 bases in length and mostpreferably 12, 13, 14, 15, 16, 17, 18, 19, or to 40 bases in length.Oligonucleotides are usually single stranded, e.g. for primers andprobes; although oligonucleotides may be double stranded, e.g. for usein the construction of a gene mutant. Oligonucleotides of the presentdisclosure can be either sense or anti-sense oligonucleotides.

As used herein, the terms “nucleic acid molecule encoding,” “DNAsequence encoding” and “DNA encoding” refer to the order or sequence ofdeoxyribonucleotides along a strand of deoxyribonucleic acid. The orderof these deoxyribonucleotides determines the order of ribonucleotidesalong the mRNA chain, and also determines the order of amino acids alongthe polypeptide (protein) chain. The DNA sequence thus codes for the RNAsequence and for the amino acid sequence.

“Expression of a gene” or “expression of a nucleic acid” meanstranscription of DNA into RNA (optionally including modification of theRNA, e.g., splicing), translation of RNA into a polypeptide (possiblyincluding subsequent post-translational modification of thepolypeptide), or both transcription and translation, as indicated by thecontext.

As used herein the term “coding region” when used in reference to astructural gene refers to the nucleotide sequences which encode theamino acids found in the nascent polypeptide as a result of translationof an mRNA molecule.

Recombinant DNA-mediated protein expression techniques are applicable tothe making of the rhGDF-5 protein. Briefly, a recombinant DNA moleculeor construct (pGDF5-T5 or pGDF5-Trc, the polynucleotide sequences ofwhich are set forth in the Sequence Listing as SEQ ID NOS: 2 and 3,respectively), coding for the gene of interest (GDF-5, thepolynucleotide sequence as set forth in Sequence Listing as SEQ ID NO:1)is prepared. Methods of preparing such DNA molecules are well known inthe art. For instance, sequences encoding the gene of interest (e.g.rhGDF-5 or GDF-5 (SEQ ID NO: 1)) can be excised from DNA using suitablerestriction enzymes. Any of a large number of available and well-knownhost cells may be used in the practice of this present disclosure. Theselection of a particular host is dependent upon a number of factorsrecognized by the art. These include, for example, compatibility withthe chosen expression vector, toxicity of the peptides encoded by theDNA molecule, rate of transformation, ease of recovery of the peptides,expression characteristics, biosafety and costs. A balance of thesefactors must be struck with the understanding that not all hosts may beequally effective for the expression of a particular DNA sequence.Within these general guidelines, useful microbial host cells in cultureinclude bacteria such as Escherichia coli sp. Modifications can be madeat the DNA level, as well. For example, the GDF-5 encoding DNA sequence(SEQ ID NO: 1) may be changed to codons more compatible with the chosenhost cell. For E. coli, optimized codons are known in the art. Codonscan be substituted to eliminate restriction sites or to include silentrestriction sites, which may aid in processing of the DNA in theselected host cell. The transformed bacterial host cell line or cellstrain is then cultured and purified. Host cells or strains may becultured under conventional fermentation conditions so that the desiredcompounds are expressed. Such fermentation conditions are well known inthe art.

The region of the vector to which the gene of interest is cloned isreferred to herein as an “insertion site.” Preferably, the gene ofinterest is rhGDF-5 or GDF-5, designated in the Sequence Listing as SEQID NO: 1.

In one embodiment, the vector comprises an Nde1 restriction site forrestriction enzyme analysis purposes.

The term “expression vector” according to the embodiment of the presentdisclosure refers to a vehicle for introducing a gene of interest into ahost cell to express the gene or a recombinant DNA molecule containing adesired coding sequence and appropriate nucleic acid sequences necessaryfor the expression of the operably linked coding sequence in aparticular host cell. Nucleic acid sequences necessary for expression inprokaryotes include a promoter, optionally an operator sequence, aribosome binding site and possibly other sequences. Expression vectorsor vectors according to the embodiment of the present disclosure includeplasmid vectors.

In one embodiment, the expression vectors of the present disclosure mayinclude regulatory promoters, examples of which may include but are notlimited to, T5, T7 and Trc promoters. The regulatory promoters of thepresent disclosure can be induced by isopropyl-β-D-thiogalactoside(IPTG).

The expression vectors of the present disclosure, which is provided forinducing high expression of a gene of interest (GDF-5 (SEQ ID NO:1)) inthe host cells, may preferably further include a resistance gene forhost cells, which is used as a selectable marker for permanentexpression of the gene in the host cells. Non-limiting examples of suchresistance genes for animal cells include those commonly used in theart, such as ampicillin-, neomycin-, kanamycin-, zeomycin- andhygromycin-resistant genes. A resistance gene, according to theembodiment of the present disclosure, is the Kanamycin-resistant(Kan^(r) gene).

According to one embodiment of the present disclosure, the term “hostcell” is used to refer to a cell which has been transformed, or iscapable of being transformed with a nucleic acid sequence and then ofexpressing a selected gene of interest (GDF-5, SEQ ID NO:1). Thus, ahost cell, as used herein, is also a transformed cell line (or strain)or a transformant.

The term “recombinant host cell” (or simply “host cell”), as usedherein, is intended to refer to a cell into which a recombinantexpression vector has been introduced. It should be understood that suchterms are intended to refer not only to the particular subject cell butto the progeny of such a cell. Because certain modifications may occurin succeeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to the parentcell, but are still included within the scope of the term “host cell” asused herein.

The term “operably linked” refers to a functional linkage between anexpression control sequence and a second nucleic acid sequence, whereinthe expression control sequence directs transcription of the nucleicacid corresponding to the second sequence.

A host cell “engineered to overexpress” a protein (or a nucleic acidencoding such protein) is a host cell, including a descendant thereof,that has been altered in such a way that higher levels of such proteinare expressed than normal, compared to the unaltered host cell. Thus,included within this category are expression of proteins foreign to thehost cell, proteins not naturally expressed by the host cell, orproteins naturally expressed by the host cell at relatively low levelsthat increase after alteration of the host cell.

In a preferred aspect, the recombinant protein of interest (rhGDF-5designated in the Sequence Listing as SEQ ID NO: 4) is expressed in theprokaryotic host cells, such as, for example E. coli host cells.Examples of prokaryotic host cell strains include, but are not limitedto DH10β, STBL2, HMS174 and recA.

The term “isolated nucleic acid” refers to a nucleic acid of the presentdisclosure that is free from at least one contaminating nucleic acidwith which it is naturally associated. A “nucleic acid” refers to a DNAor RNA sequence, optionally including artificial bases or base analogs.

The term “identity” (or “percent identical”) is a measure of the percentof identical matches between the smaller of two or more sequences withgap alignments (if any) addressed by a particular mathematical model orcomputer program (i.e., “algorithms”). The term “similarity” is arelated concept but, in contrast to “identity”, includes both identicalmatches and conservative substitution matches. Identity and similarityof related nucleic acid molecules and polypeptides can be readilycalculated by known methods. Preferred methods to determine identityand/or similarity are designed to give the largest match between thesequences tested. Methods to determine identity and similarity aredescribed in publicly available computer programs. Exemplary computerprogram methods to determine identity and similarity between twosequences include, but are not limited to, the GCG program package,including GAP (Devereux et al., Nucl. Acids. Res. 12: 387, 1984;Genetics Computer Group, University of Wisconsin, Madison, Wis.),BLASTP, BLASTN, and FASTA (Altschul et al., J. Mol. Biol. 215:403-410,1990)). The BLASTX program is publicly available from the NationalCenter for Biotechnology Information (NCBI) and other sources (BLASTManual, Altschul et al. NCB/NLM/NIH Bethesda, Md. 20894; Altschul etal., supra). The well-known Smith-Waterman algorithm may also be used todetermine identity. Preferred parameters for a polypeptide sequencecomparison include the following: Algorithm: Needleman et al., J. Mol.Biol. 48: 443-53 (1970); Comparison matrix: BLOSUM 62 from Henikoff etal., Proc. Natl. Acad. Sci. USA 89:10915-19 (1992); Gap Penalty: 12, GapLength Penalty: 4; Threshold of Similarity: 0. The GAP program is usefulwith the above parameters (along with no penalty for end gaps).Preferred parameters for nucleic acid molecule sequence comparisonsinclude the following: Algorithm: Needleman et al., J. Mol. Biol.,48:443-53 (1970); Comparison matrix: matches=+10, mismatch=0, GapPenalty: 50, Gap Length Penalty: 3. The GAP program is also useful withthe above parameters. Other exemplary algorithms, gap opening penalties,gap extension penalties, comparison matrices, thresholds of similarity,etc. may be used by those of skill in the art.

The phrase “stringent hybridization conditions” refers to conditionsunder which a probe will hybridize to its target subsequence, typicallyin a complex mixture of nucleic acid, but to no other sequences.Hybridization stringency is principally determined by temperature, ionicstrength, and the concentration of denaturing agents such as formamide.Examples of “highly stringent conditions” for hybridization and washingare 0.015M sodium chloride, 0.0015M sodium citrate at 65-68° C. or0.015M sodium chloride, 0.0015M sodium citrate, and 50% formamide at 42°C. See Sambrook, Fritsch & Maniatis, Molecular Cloning: A LaboratoryManual, 2nd Ed., Cold Spring Harbor Laboratory, (Cold Spring Harbor,N.Y. 1989); and Anderson et al., Nucleic Acid Hybridization:Hybridization: a practical approach, Ch. 4, IRL Press Limited (Oxford,England) (1999). Examples of typical “moderately stringent” conditionsare 0.015M sodium chloride, 0.0015M sodium citrate at 50-65° C. or0.015M sodium chloride, 0.0015M sodium citrate, and 20% formamide at37-50° C. By way of example, a “moderately stringent” condition of 50°C. in 0.015 M sodium ion will allow about a 21% mismatch.

It will be appreciated by those skilled in the art that there is noabsolute distinction between “highly” and “moderately” stringentconditions. For example, at 0.015M sodium ion (no formamide), themelting temperature of perfectly matched long DNA is about 71° C. With awash at 65° C. (at the same ionic strength), this would allow forapproximately a 6% mismatch. To capture more distantly relatedsequences, one skilled in the art can simply lower the temperature orraise the ionic strength.

The nucleotide and amino acid sequences of pGDF5-T5 and pGDF5-Trc areset forth in SEQ ID NOS: 2 and 3 and SEQ ID NO: 4, respectively.

The term “rhGDF-5 or GDF-5” as used herein refers to human growth anddifferentiation factor-5 (the polypeptide of SEQ ID NO:4 encoded by thepolynucleotide of SEQ. ID NO: 1) thereof, or a biologically activefragment, variant, analog, or derivative of the human GDF-5 protein.Exemplary analogs retain 65% or higher amino acid identity to the parentsequence, or 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% or higher identity.

As used herein, the term “rhGDF-5 or GDF-5 (pGDF5-T5 or pGDF5-Trcnucleic acid” or “rhGDF-5 or GDF-5 (pGDF5-T5 or pGDF5-Trc)polynucleotide” refers to a nucleic acid that encodes a polypeptidehaving an amino acid sequence as set forth in SEQ ID NO:4, including anucleotide sequence as set forth in SEQ ID NO: 1, or nucleic acidscomprising nucleotide sequences that are at least about 65%, 70%, 75%,80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical thereto, ornucleic acids which hybridize under moderately or highly stringentconditions as defined herein with the complement of SEQ ID NO: 1 or anyother orthologs of the nucleotide sequence of SEQ ID NO: 1.

The terms “polypeptide” and “protein” are used interchangeably herein.

Overexpression, as described herein, encompasses activating (or causingto be expressed) a gene which is normally silent (unexpressed) in thehost cell as obtained, as well as increasing the expression of a genewhich is not expressed at physiologically significant levels in the cellas obtained.

The present disclosure includes methods and compositions for theproduction of rhGDF-5 using the T5 or Trc promoter in the production ofrhGDF-5 for therapeutic applications. The rh-GDF-S can be easilyproduced in large scale quantities in cost-effective, and time-savingmanner.

According to the embodiment of the present disclosure, the term“isolated protein” comprises rhGDF-5 or GDF5 protein. In one embodiment,the protein comprises rhGDF-5 or GDF5 having the amino acid sequence setforth in SEQ ID NO:4 and variants and derivatives of this protein, whichretain the activity of the polypeptide of SEQ ID NO: 4. In oneembodiment, the protein comprises a polypeptide having at least about80% identity, at least about 85% identity, at least about 90% identity,at least about 95% identity, at least about 98% identity, or at leastabout 99% identity to the amino acid sequence set forth in SEQ ID NO: 4.

Stable production of proteins, including biologics, can be accomplishedby transfecting host cells with vectors containing DNA that encodes theprotein. Maintenance of the vector in the cell line can be achievedthrough a variety of means

With the evolving importance of therapeutic proteins, i.e., biologics,efforts must be made to optimize protein production, while improvingefficiency of the overall production process. Thus, improvements inefficiency must be weighed against the protein production capacity ofthe vector. There is a need for better expression systems that provideefficient cloning options, as well as high levels of the desired proteinproduct. It would be advantageous to decrease the number of cloningsteps involved in the production of biologics to improve timerequirements and minimize cost. It would also be advantageous to providevectors that provide adequate protein production for both small andlarge scale cell cultures.

After the expression vector is introduced into the cells, thetransfected cells are cultured under conditions favoring expression ofthe protein, which may be recovered from the culture using standardtechniques identified below by contacting the protein with a substrate(e.g., resin, glass, plastic, cellulose, etc.) that is capable ofimmobilizing the protein.

The substrate comprises at least one surface on which to capture theGDF-5 protein. The substrate may comprise a ceramic substance, a glass,a metal, a crystalline material, a plastic, a polymer or co-polymer, anycombinations thereof, or a coating of one material on another. Suchsubstrates include for example, but are not limited to, (semi) noblemetals such as gold or silver; glass materials such as soda-lime glass,pyrex glass, vycor glass, quartz glass; resins, cellulose, metallic ornon-metallic oxides; silicon, monoammonium phosphate, and other suchcrystalline materials; transition metals; plastics or polymers,including dendritic polymers, such as poly(vinyl chloride), poly(vinylalcohol), poly(methyl methacrylate), poly(vinyl acetate-maleicanhydride), poly(dimethylsiloxane) monomethacrylate, polystyrenes,polypropylene, polyethyleneimine; copolymers such as poly(vinylacetate-co-maleic anhydride), poly(styrene-co-maleic anhydride),poly(ethylene-co-acrylic acid) or derivatives of these or the like.

The substrate may take a variety of configurations ranging from simpleto complex, depending on its intended use. Thus, the substrate couldhave an overall slide or plate configuration, such as a rectangular ordisc configuration or be part of a column.

Proteins (e.g., rh-GDF-5) and/or fragments thereof can be purified fromany suitable expression system. If desired, the protein may be purifiedto substantial purity by standard techniques, including selectiveprecipitation with such substances as ammonium sulfate; columnchromatography, immunopurification methods, and others (see, e.g.,Scopes, Protein Purification: Principles and Practice (1982); and U.S.Pat. No. 4,673,641. However, in the practice, purified or partiallypurified proteins are not required for either treatment with a modifyingagent or generation of antibodies. If purification of the aconformationally trapped protein is desired, the presence of themodifying agent may be used to aid in the purification by allowing askilled artisan to follow the location of the conformationally trappedprotein during various purification steps such as those described below.

Recombinant proteins (e.g., rh-GDF-5) can be expressed by transformedbacteria in large amounts, typically after promoter induction; butexpression can be constitutive. Promoter induction with a T5 or a Trcpromoter is one example of an inducible promoter system. Bacteria aregrown. Fresh or frozen bacteria cells are used for isolation of protein.Proteins (e.g., rh-GDF-5) expressed in bacteria may form insolubleaggregates (“inclusion bodies”). Several protocols are suitable forpurification of the expressed proteins from inclusion bodies. Forexample, purification of inclusion bodies typically involves theextraction, separation and/or purification of inclusion bodies bydisruption of bacterial cells. The cell suspension can be lysed using2-3 passages through a French Press; homogenized using, for example, aPolytron (Brinkman Instruments); disrupted enzymatically, e.g., by usinglysozyme; or sonicated on ice. Alternate methods of lysing bacteria areapparent to those of skill in the art (see, Sambrook et al., MolecularCloning, A Laboratory Manual (2nd ed. 1989); Kriegler, Gene Transfer andExpression: A Laboratory Manual (1990); and Current Protocols inMolecular Biology (Ausubel et al., eds., 1994)).

If necessary, the inclusion bodies are solubilized, and the lysed cellsuspension is typically centrifuged and/or filtered to remove unwantedinsoluble matter. Proteins that formed the inclusion bodies may berenatured by dilution or dialysis with a compatible buffer. Suitablesolvents include, but are not limited to urea (from about 4 M to about 8M), formamide (at least about 80%, volume/volume basis), and guanidinehydrochloride (from about 4 M to about 8 M). Some solvents which arecapable of solubilizing aggregate-forming proteins, for example SDS(sodium dodecyl sulfate), 70% formic acid, are inappropriate for use inthis procedure due to the possibility of irreversible denaturation ofthe proteins, accompanied by a lack of immunogenicity and/or activity.Although guanidine hydrochloride and similar agents are denaturants,this denaturation is not irreversible and renaturation may occur uponremoval (by dialysis, for example) or dilution of the denaturant,allowing re-formation of immunologically and/or biologically activeprotein. Other suitable buffers are known to those skilled in the art.One of skill in the art will recognize that optimal conditions forrenaturation must be chosen for each protein. For example, if a proteinis soluble only at low pH, renaturation can be done at low pH.Renaturation conditions can thus be adjusted for proteins with differentsolubility characteristics i.e., proteins that are soluble at neutral pHcan be renatured at neutral pH.

The expressed protein (e.g., rh-GDF-5) is separated from other bacterialproteins by standard separation techniques, such as for example,solubility fractionation, size differential filtration, columnchromatography, or the like. Solubility fractionation involves aninitial salt fractionation can separate many of the unwanted host cellproteins (or proteins derived from the cell culture media) from therecombinant protein of interest. In some embodiments, ammonium sulfatecan be used. Ammonium sulfate precipitates proteins by effectivelyreducing the amount of water in the protein mixture. Proteins thenprecipitate on the basis of their solubility. The more hydrophobic aprotein is, the more likely it is to precipitate at lower ammoniumsulfate concentrations. A typical protocol includes adding saturatedammonium sulfate to a protein solution so that the resultant ammoniumsulfate concentration is between 20-30%. This concentration willprecipitate the most hydrophobic of proteins. The precipitate is thendiscarded (unless the protein of interest is hydrophobic) and ammoniumsulfate is added to the supernatant to a concentration known toprecipitate the protein of interest. The precipitate is then solubilizedin buffer and the excess salt removed if necessary, either throughdialysis or diafiltration. Other methods that rely on solubility ofproteins, such as cold ethanol precipitation, are well known to those ofskill in the art and can be used to fractionate complex proteinmixtures.

Size differential filtration involves using the molecular weight of agiven protein to isolate it from proteins of greater and lesser sizeusing ultrafiltration through membranes of different pore size (forexample, Amicon or Millipore membranes). As a first step, the proteinmixture is ultrafiltered through a membrane with a pore size that has alower molecular weight cut-off than the molecular weight of the proteinof interest. The retentate of the ultrafiltration is then ultrafilteredagainst a membrane with a molecular cut off greater than the molecularweight of the protein of interest. The recombinant protein will passthrough the membrane into the filtrate. The filtrate can then bechromatographed as described below.

Column chromatography involves separating a protein from other proteinson the basis of its size, net surface charge, hydrophobicity, andaffinity for ligands. In addition, antibodies raised against proteinscan be conjugated to column matrices and the proteins immunopurified.All of these methods are well known in the art. It will be apparent toone of skill that chromatographic techniques can be performed at anyscale and using equipment from many different manufacturers (e.g.,Pharmacia Biotech). In one embodiment, the column comprises a resin thatbinds the GDF-5 protein in order to isolate the rh-GDF-5 protein. Theprotein can be washed from the resin and washed with a buffer to refoldthe GDF-5 protein.

In some embodiments, the rh-GDF-5 can be isolated on the substrate indifferent conformational states. A modifying agent can be contacted witha protein of interest to fix the protein in a specific conformationalstate. The interaction of the modifying agent may be covalent ornon-covalent and may be reversible or essentially irreversible.Functional groups on proteins that may serve as sites for attachment ofmodifying agents include sulfhydryl, amino, and carboxyl groups found onthe side chains of various amino acids. Furthermore, other features of aprotein such as charge, hydrophobicity, or hydrogen bonding potential,among others, may serve as a point of association between a modifyingagent and a protein. Examples of trapping of conformational states usingmodifying agents such as those which react with sulfhydryl groups may befound in: Erlanson et al., Annual Review of Biophys. Biomol. Struct.,33:199-223 (2004), Erlanson, et al., Curr. Opin. Chem. Biol., 8: 399-406(2004), Erlanson et al., J. Med. Chem., 47: 3463-3482 (2004), Hardy etal., Proc. Nat'l. Acd. Sci., 34:12461-12466 (2004); Buck and Wells,Proc. Nat'l. Acd. Sci., 102: 2719-2724 (2005); Scheer et al., Proc.Nat'l. Acd. Sci., 103: 7595-7600 (2006), which are incorporated byreference in their entirety.

Antibodies can be used to isolate the rh-GDF-5 in differentconformational states. Methods of producing polyclonal and monoclonalantibodies that react specifically with proteins are known to those ofskill in the art and can be readily adapted to generate conformationspecific antibodies by using the conformationally trapped proteinsdescribed above as antigens (see, e.g., Coligan, Current Protocols inImmunology (1991); Harlow & Lane, supra; Goding, Monoclonal Antibodies:Principles and Practice (2d ed. 1986); and Kohler & Milstein, Nature256:495-497 (1975). Such techniques include antibody preparation byselection of antibodies from libraries of recombinant antibodies inphage or similar vectors, as well as preparation of polyclonal andmonoclonal antibodies by immunizing rabbits or mice (see, e.g., Huse etal., Science 246:1275-1281 (1989); Ward et al., Nature 341:544-546(1989)).

Methods of production of polyclonal antibodies are known to those ofskill in the art. An inbred strain of mice (e.g., BALB/C mice) orrabbits is immunized with the protein (i.e., immunogen) using a standardadjuvant, such as Freund's adjuvant, and a standard immunizationprotocol. The animal's immune response to the immunogen preparation ismonitored by taking test bleeds and determining the titer of reactivityto the protein. When appropriately high titers of antibody to theimmunogen are obtained, blood is collected from the animal and antiseraare prepared. Further fractionation of the antisera to enrich forantibodies reactive to the protein can be done if desired (see, Harlow &Lane, supra).

Monoclonal antibodies may be obtained by various techniques familiar tothose skilled in the art. Briefly, spleen cells from an animal immunizedwith a desired antigen are immortalized, commonly by fusion with amyeloma cell (see, Kohler & Milstein, Eur. J. Immunol. 6:511-519(1976)). Alternative methods of immortalization include transformationwith Epstein Barr Virus, oncogenes, or retroviruses, or other methodswell known in the art. Colonies arising from single immortalized cellsare screened for production of antibodies of the desired specificity andaffinity for the antigen, and yield of the monoclonal antibodiesproduced by such cells may be enhanced by various techniques, includinginjection into the peritoneal cavity of a vertebrate host.Alternatively, one may isolate DNA sequences which encode a monoclonalantibody or a binding fragment thereof by screening a DNA library fromhuman B cells according to the general protocol outlined by Huse, etal., Science 246:1275-1281 (1989).

Monoclonal antibodies and polyclonal sera are collected and titeredagainst the immunogen protein (e.g., GDF-5) in an immunoassay, forexample, a solid phase immunoassay with the immunogen immobilized on asolid support. Typically, polyclonal antisera with a titer of 10⁴ orgreater are selected and tested for their cross reactivity againstnon-conformationally trapped proteins or proteins trapped in aconformation different from that used to raise the antibody. Specificpolyclonal antisera and monoclonal antibodies will usually bind with aKd of at least about 0.1 mM, more usually at least about 1 μM,preferably at least about 0.1 μM or better, and most preferably, 0.01 μMor better.

Once antibodies specific to a particular conformational state areavailable, the antibodies can be labeled with a suitable label (e.g.,dye, fluorophore, tage, etc.) to identify the particular conformationalstate of the GDF-5 protein or to identify the GDF-5 protein if needed.The antibodies used to identify the GDF-5 protein may optionally becovalently or non-covalently linked to a detectable label. Detectablelabels suitable for such use include any composition detectable byspectroscopic, photochemical, biochemical, immunochemical, electrical,optical or chemical means. Useful include magnetic beads (e.g.DYNABEADS), fluorescent dyes (e.g., Alexa Fluor 350, Alexa Fluor 405,Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 500, Alexa Fluor 514,Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568,Alexa Fluor 594, Alexa Fluor 610, Alexa Fluor 633, Alexa Fluor 635,Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700 andAlexa Fluor 750 dyes, fluorescein isothiocyanate, Texas red, rhodamine,green fluorescent protein, and the like), radiolabels (e.g., .sup.³H,.sup.¹²5I, .sup.³⁵S, .sup.¹⁴C, or .sup.³²P), enzymes (e.g., horse radishperoxidase, alkaline phosphatase and others commonly used in an ELISA),and colorimetric labels such as colloidal gold or colored glass orplastic (e.g. polystyrene, polypropylene, latex, etc.) beads.

Means of detecting such labels are well known to those of skill in theart. Thus, for example, radiolabels may be detected using photographicfilm or scintillation counters, fluorescent markers may be detectedusing a photodetector to detect emitted illumination. Enzymatic labelsare typically detected by providing the enzyme with a substrate anddetecting the reaction product produced by the action of the enzyme onthe substrate, and colorimetric labels are detected by simplyvisualizing the colored label.

Conformation specific antibodies may be used in virtually any assayformat that employs antibodies to detect antigens. Design of theimmunoassays is subject to a great deal of variation, and many formatsare known in the art. Protocols may, for example, use solid supports, orimmunoprecipitation. Many assays involve the use of labeled antibody orpolypeptide; the labels may be, for example, enzymatic, fluorescent,chemiluminescent, radioactive, or dye molecules, as discussed in detailabove. Assays which amplify the signals from the immune complex are alsoknown; examples of which are assays which utilize biotin and avidin, andenzyme-labeled and mediated immunoassays, such as ELISA assays.

In one embodiment, the expressed recombinant rhGDF-5 protein, accordingto the embodiment of the present disclosure, can be collected frompGDF5-T5 or pGDF5-Trc transformed host cell lysates (from strains DH10β,STBL2, HMS174 or RecA). The supernatant (soluble fraction) and pellet(insoluble fraction containing inclusion bodies) can be separated bycentrifugation. The pellet may then be collected and disrupted orhomogenized to release the inclusion bodies from the bacterial cells.Host cell disruption or homogenization may be performed using well knowntechniques including, but not limited to, enzymatic cell disruption,sonication, dounce homogenization or high pressure release disruption.In one embodiment, the techniques disclosed are used to disrupt thepGDF5-T5- or pGDF5-Trc-transformed E. coli cells to release theinclusion bodies of rhGDF-5 protein.

In one embodiment, after cell disruption, the inclusion bodies may thenbe subjected to solubilization using suitable denaturing agents known inthe art. The denaturing agents may be urea or guanidine hydrochloride.The recombinant rhGDF-5 protein can be recovered and purified from theresulting solution by any of a number well known in the art, includingbut are not limited to, using ion-exchange chromatography, ammoniumsulfate or ethanol precipitation, acid or base extraction, columnchromatography, affinity column chromatography, anion or cation exchangechromatography, phosphocellulose chromatography, hydrophobic interactionchromatography, hydroxylapatite chromatography, lectin chromatography,gel electrophoresis and the like. Protein refolding steps can be used,as desired, in making correctly folded mature proteins. High performanceliquid chromatography (HPLC), affinity chromatography or other suitablemethods can be employed in final purification steps where high purity isdesired. Once purified, partially or to homogeneity, as desired, therhGDF-5 proteins are optionally used for a wide variety of utilities,including but not limited to, as assay components, therapeutics(biologics), prophylaxis, diagnostics, research reagents, and/or asimmunogens for antibody production.

In addition to other references noted herein, a variety ofpurification/protein folding methods are well known in the art,including, but not limited to, those set forth in R. Scopes, ProteinPurification, Springer-Verlag, N.Y. (1982); Deutscher, Methods inEnzymology Vol. 182: Guide to Protein Purification, Academic Press, Inc.N.Y. (1990); Sandana (1997) Bioseparation of Proteins, Academic Press,Inc.; Bollag et al. (1996) Protein Methods, 2nd Edition Wiley-Liss, NY;Walker (1996) The Protein Protocols Handbook Humana Press, NJ, Harrisand Angal (1990) Protein Purification Applications: A Practical ApproachIRL Press at Oxford, Oxford, England; Harris and Angal ProteinPurification Methods: A Practical Approach IRL Press at Oxford, Oxford,England; Scopes (1993) Protein Purification: Principles and Practice 3rdEdition Springer Verlag, NY; Janson and Ryden (1998) ProteinPurification: Principles, High Resolution Methods and Applications,Second Edition Wiley-VCH, NY; and Walker (1998) Protein Protocols onCD-ROM Humana Press, NJ; and the references cited therein.

As used herein, the term “exponential growth” refers to that portion ofthe cellular growth cycle between the lag phase and the stationary phasewhen cells are doubling at a logarithmic rate. The term “exponentialgrowth” is also meant to encompass the late lag phase (i.e., the earlystationary phase) which occurs between the logarithmic growth phase andstationary phase, when the cell growth rate is slowing, and thereforencompasses an extended exponential growth phase. Therefore, “stationaryphase” refers to horizontal growth, i.e., when the cells haveessentially stopped dividing and have reached a quiescent stage withrespect to cell doubling.

As in conventional fermentation processes, it is usually desirable toobtain as high a rate of cell growth in as high a density of bacterialcell culture as possible, to maximize the amount of bacterial biomassproduced per unit of time. “Biomass,” as utilized herein and withoutalteration from its conventional meaning, refers to the mass and/oraccumulating mass of host bacterial cells or transforming bacteria cellsresulting from the cultivation of such cells using a variety oftechniques, e.g., cultivating such cells in defined or semi-definedmedia containing additional ingredients that may enhance or increasebacterial growth rate or biomass.

According to the embodiment of the present disclosure, fermentationprocesses have been developed to maximize the yield of pGDF5-T5 andpGDF5-Trc DNAs from large scale cultures of transformed host cells andto optimize recombinant hGDF-5 protein production. The fermentationprocesses includes optimizing the plasmid yield such that the supply ofmetabolites essential for growth is adequate to permit growth to a highbiomass, but is not in excess so as to inhibit such growth.

According to one embodiment, host cells transformed with an expressionvector that includes a pGDF5-Trc or pGDF5-T5 DNA are cultured in a highcell density medium that are modified based on its response to proteinexpression and biomass yield. For example, a defined media mayadditionally include ingredients such as sodium molybdate (ranges fromabout 5 to about 10 mg/L), magnesium sulfate heptahydrate (ranges fromabout 2 to about 6 mM), sodium chloride (ranges from 0 to about 4 g/L),EDTA (ranges from 0 to about 400 mg/L), MOPS (ranges from 0 to about 100mM), amino acid supplement including L-methionine (ranges from 0 toabout 10 ml/L) and vitamin supplement (folic acid, pyridoxine, andbiotin; ranges from 0 to about 10 ml/L). Alternatively, as semi-defined(complex) media may include, in addition to what was in the definedmedia, yeast extract and tryptone (animal-derived). Both yeast extractand tryptone range from 0 to about 0.4% w/v. Also included were sodiummolybdate, magnesium sulfate, sodium chloride, EDTA, MOPS(3-[N-morpholino] propane-sulfonic acid), amino acids (includingL-methionine), and vitamins (folic acid, pyrodoxine, and biotin).

A type of fermentation according to the embodiment of the presentdisclosure is fed-batch fermentation, in which the cell growth rate iscontrolled by the addition of nutrients to the culture during cellgrowth. As used herein, “fed-batch fermentation” refers to a cellculture process in which the growth rate is controlled by carefullymonitored additions of metabolites to the culture during fermentation.Fed-batch fermentation according to the present disclosure permits thecell culture to reach a higher biomass. The key to fed-batchfermentation is supplying substrate at a rate such that it is completelyconsumed. As a result, residual substrate concentration is approximatelyzero and maximum conversion of substrate is obtained. Metabolic overflowfrom excess substrate is avoided, reducing the formation of inhibitoryacetate. Fed-batch fermentation starts with a batch phase. Cells areinoculated into an initial volume of medium that contains allnon-limiting nutrients and an initial concentration of the limitingsubstrate. Controlled feeding of the limiting nutrient begins once thecells have consumed the initial amount of substrate. One of the simplestand most effective feeding strategies is exponential feeding. Thismethod allows the culture to grow at a predetermined rate less than thepopulation doubling time, as expressed herein, as mu (μ)_(max) withoutthe need of feedback control. The fermentation begins with a batch modecontaining a non-inhibitory concentration of substrate. The cells growat mu (μ)_(max) until the substrate is exhausted, at which point thenutrient feeding begins.

The DO-stat and pH-stat methods are fairly easy to implement since moststandard fermentor systems include dissolved oxygen and pH monitoring.Trends in dissolved oxygen (DO) and pH can indicate whether substrate isavailable to the cells. Exhaustion of substrate causes decreased oxygenuptake and the DO concentration in the medium rises. The pH also risesdue to consumption of metabolic acids. Feeding is triggered when DO orpH rises above a set threshold. The growth rate can be adjusted bychanging the DO or pH threshold value.

EXAMPLES

Reference will now be made in detail to certain embodiments of thepresent disclosure. While the present disclosure will be described inconjunction with the illustrated embodiments, it will be understood thatthey are not intended to limit the present disclosure to thoseembodiments. On the contrary, the present disclosure is intended tocover all alternatives, modifications, and equivalents that may beincluded within the present disclosure as defined by the appendedclaims. The headings below are not meant to limit the disclosure in anyway; embodiments under any one heading may be used in conjunction withembodiments under any other heading.

Nucleotide and amino acid sequences are referred to herein by a sequenceidentifier number (SEQ ID NOS:1 to 39). A sequence listing is providedat the end of the specification.

GDF5-T5 and GDF5-Trc Plasmid/Expression Vector Construction

Two plasmid expression vectors (pGDF5-T5 and pGDF5-Trc) were engineeredfor the purpose of expressing a recombinant human growth anddifferentiation factor-5 (rhGDF-5) of about 13.5 kDa in selectedprokaryotic host cell strains. Plasmid maps for both plasmids areprovided in FIGS. 1A and 1B. The sizes of pGDF5-T5 and pGDF5-Trc are4299 and 4403 bp, respectively. The complete nucleotide sequence of thepGDF5-T5 and pGDF5-Trc is designated in the Sequence Listing as SEQ IDNOS: 2 and 3, respectively.

To do this, a starting IP constraint-free plasmid, pJExpress-401(DNA2.0, Menlo Park, Calif. having either a T5 or a Trc promoter (bothIPTG-inducible) was employed. Some of the features of pJExpress-401include a pUC origin, a Kanamycin selective marker gene (Kan^(R)) gene,either a T5 or a Trc promoter (both IPTG(isopropylthio-(3-galactoside)-inducible) and an Nde-1 restriction site.The gene of interest (gene insert) is a codon-optimized human GDF-5(rhGDF-5) cDNA having a size of 528 bp designated in the SequenceListing as (SEQ ID NO:1). The reverse complimentary coding sequence ofthe insert sequence, as underlined is provided in the sequence listingas SEQ ID NO:7.

Plasmid DNA Analysis

As indicated above, the complete DNA sequences for pGDF5-T5 andpGDF5-Trc are provided in the sequence listing as SEQ ID NOS:2 and 3,respectively. The theoretical protein sequence encoded by pGDF5-Trc isdenoted herein as SEQ ID. NO. 4. Sequencing was performed on pGDF5-T5and pGDF5-Trc with a minimum of 2× coverage over the backbone and 4×coverage over the gene of interest (GDF-5 DNA insert). The samples wererun on the ABI 3130x1 genetic analyzer and analyzed using ABI'sSequencing Analysis software version 5.3.1. The sequences were editedusing Sequencher™ version 5.0. The sequences obtained were assembledinto contiguous sequence files. The consensus sequence (MD1.seqcorresponding to SEQ ID NO:3) was compared with the correspondingexpected reference sequence file (MD1.txt corresponding to SEQ ID NO:6).The sequence file comparisons and additional data are discussedhereinbelow.

Plasmid DNA and Sequencing Primers Preparation and Purification:

Prior sequencing, the concentration of purified plasmid GDF5-Trc andGDF5-T5 DNA (pGDF5-Trc and pGDF5-T5) was determined using the Smartspec™3000 spectrophotometer. Sequencing Primers (oligonucleotides) weredesigned accordingly and the following primers, as well as thepolynucleotides and polypeptides according to the embodiments of thepresent disclosure, are listed on Table 1. The sequencing primers wereutilized to sequence pGDF5-Trc.

TABLE 1 SEQ ID NO. Description Sequence Species/Type Length StartDirection Tm % GC 1 GDF-5 DNA See Sequence Listing human/DNA 528insert-insert DNA from pGDF5-Trc plasmid 2 complete See Sequence Listinghuman/DNA 4299 sequence of pGDF5-T5 DNA 3 complete See Sequence Listinghuman/DNA 4403 sequence of pGDF5-Trc DNA 4 theoretical See SequenceListing human/protein 125 amino acid sequence of rhGDF-5 protein 5GDF5-CofA See Sequence Listing human/protein 120 from Prospec-TanyTechnoGene Ltd 6 MD1.txt See Sequence Listing human/DNA 4405reference GDF-5 sequence for Sequencing 7 Reverse See Sequence Listinghuman/DNA 528 complementary strand insert sequence from pGDF5-Trcplasmid 8 MDP1.1SF1-A CTATCATGCCATACCGCGAAA artificial sequence 21 35Forward 60 48 9 MDP1.2SF1-A GCCAGCCATTACGCTCGTC artificial sequence 19382 Forward 60 63 10 MDP1.3SF1-A CGCTACCTTTGCCATGTTTCA artificialsequence 21 721 Forward 60 48 11 MDP1.4SF1-A TAATCGCGGCCTCGACGartificial sequence 17 857 Forward 60 65 12 MDP1.5SF1-ACCTGACCCCATGCCGAA artificial sequence 17 1105 Forward 60 65 13MDP1.6SF1-A AGTTAGCGACAGCCGCAGC artificial sequence 19 1328 Forward 6063 14 MDP1.7SF1-A ATGGCTACGCAGCGGAAAC artificial sequence 19 1516Forward 60 58 15 MDP1.8SF1-A GCGGCATATGTTTTACCTCCTG artificial sequence22 1686 Forward 59 50 16 MDP1.9SF1-A AGCTCGTAATTGTTATCCGCTCA artificialsequence 23 1812 Forward 59 43 17 MDP1.10SF1-A CAAGCAAAGTGACAGGCGCartificial sequence 19 2214 Forward 59 58 18 MDP1.11SF1-AGGCGGTAATACGGTTATCCACA artificial sequence 22 2542 Forward 60 50 19MDP1.12SF1-A TGCGCCTTATCCGGTAACTATC artificial sequence 22 2933 Forward59 50 20 MDP1.13SF1-A TTTTGGTCATGAGTCACTGC artificial sequence 20 3311Forward 53 45 21 MDP1.14SF1-A GGAACGATGCCCTCATTCAG artificial sequence20 3691 Forward 59 55 22 MDP1.15SF1-A CCAGCGGATAGTTAATGATCAGC artificialsequence 23 4022 Forward 59 48 23 MDP1.16SF1-A CCGGCATACTCTGCGACATCartificial sequence 20 4366 Forward 60 60 24 MDP1.17SR1-AGATGTCGCAGAGTATGCCGG artificial sequence 20 21 Reverse 60 60 25MDP1.18SR1-A CATTAACTATCCGCTGGATGACC artificial sequence 23 368 Reverse59 48 26 MDP1.19SR1-A GCCAACGATCAGATGGCG artificial sequence 18 732Reverse 60 61 27 MDP1.20SR1-A TGACCAAAATCCCTTAACGTGAGT artificialsequence 24 1087 Reverse 60 42 28 MDP1.21SR1-A GATAGTTACCGGATAAGGCGCAartificial sequence 22 1452 Reverse 59 50 29 MDP1.22SR1-ACCTGCGTTATCCCCTGATTCT artificial sequence 21 1823 Reverse 59 52 30MDP1.23SR1-A AAACGACGGCCAGTCTTAAGCT artificial sequence 22 2029 Reverse60 50 31 MDP1.24SR1-A AACGTAAAAACCCGCTTCGG artificial sequence 20 2099Reverse 60 50 32 MDP1.25SR1-A CGCCTGTCACTTTGCTTGATA artificial sequence21 2175 Reverse 58 48 33 MDP1.26SR1-A TGAGCGGATAACAATTACGAGCT artificialsequence 23 2572 Reverse 59 43 34 MDP1.27SR1-A TTGCTCCCGTAAAGCCCTGartificial sequence 19 2767 Reverse 60 58 35 MDP1.28SR1-ACCCGATCTCTATTCTGTTCATCG artificial sequence 23 2986 Reverse 59 48 36MDP1.29SR1-A TACGGCGTTTCACTTCTGAGTTC artificial sequence 23 3265 Reverse59 48 37 MDP1.30SR1-A GGTGCGACAATCTATCGCTTG artificial sequence 21 3613Reverse 59 52 38 MDP1.31SR1-A GATCGCGTATTTCGCCTCG artificial sequence 193934 Reverse 60 58 39 MDP1.32SR1-A CTGCCTCGGTGAGTTTTCTCC artificialsequence 21 4206 Reverse 60 57

The sequencing primers were diluted to a final concentration of 1.6pmol/pL and the cycle sequencing reactions were setup in a 96-wellplates. The cycle sequencing plates were then loaded onto the ABIVeriti® Thermal Cycler for a cycle sequencing run based on the followingcycling conditions (see Table 2):

TABLE 2 Cycling Conditions: 98° C. 5 minutes 30 cycles: 96° C., 30seconds 50° C., 10 seconds 62° C., 4 minutes Hold:  4° C., 00

Sample purification (dye terminator removal) occurred after the cyclesequencing run. The samples were purified using Qiagen® Dye Ex 2.0 SpinKit. The samples were eluted with Hi-Di Formamide and transferred to96-well plates. The 96-well plates were denatured at 95° C. for 2minutes in the ABI Veriti® Thermal Cycler.

Sequencing Analysis:

The 96-well sequencing plates were run on the ABI 3130x1 GeneticAnalyzer using ABI's Data Collection Software version 3.0. Thesequencing data (electropherograms) were analyzed using ABI SequenceAnalysis software version 5.3.1. The sequences were edited and assembledinto contiguous sequence files using Sequencher™ Version 5.0. Thefollowing ambiguity codes may appear in the sequence files:

TABLE 3 Symbol: Meaning: 1 Probable C 2 Probable T 3 Probable A 4Probable G R A or G Y C or T M A or C K G or T W A or T S G or C H A orC or T B G or T or C V G or C or A D G or T or A N A, C, G, or TDNA Sequencing Analysis Summary:

Of the 4405 bps provided, 4403 bps was sequenced. The plasmid pGDF5-TrcDNA, designated herein as SEQ ID NO:3 was sequenced in full with aminimum of 2× coverage over entire plasmid and 4× coverage over theinsert (bp 1324-4851). The insert sequence was conforming and there were3 discrepancies found outside the coding region (DNA insert): (i) one(1) ambiguity (Y at consensus position bp: 116) and 2 discrepancies (ii)two (2) deletions in supplied sequence (bases that appear in sequence(MD1.txt of SEQ ID NO:6) but not in the sequenced data (MD1.seq of SEQID NO:3)) at consensus positions bp: 86 and 87). Protein alignmentbetween the theoretical amino sequence encoded by pGDF5-Trc shows thatit is about 99% identical to the amino acid sequence of acommercially-known recombinant human GDF-5 protein (Catalog No. CYT-442;Prospec-TanyTechnoGene Ltd, Rehovot, Israel). See FIG. 1C and SequenceID NOS:4 and 5, respectively).

pGDF5-T5 and pGDF5-Trc Transformation

Four Escherichia coli bacteria host cell lines were selected forpGDF5-T5 and pGDF5-Trc transformation: (1) DH10β (genotype: F-, mcrAΔ(mrr-hsdRMS-mcrBC) φ80lacZΔM15 ΔlacX74 recA1 endA1 araD139 Δ (ara,leu)7697 galU galK λ-rpsL nupG/pMON14272/MON7124); (2) STBL2 (genotype:F-, mcrA Δ(mcrBC-hsdRMS-mrr) recA1 endA1lon gyrA96 thi supE44 relA1λ-Δ(lac-proAB); (3) HMS174 (genotype: F-, recA1 hsdR(rK12-mK12+)(RifR);and (4) recA (genotype: recA1819 complete gene deletion).

A. Preparation of Media and Agar Plates

“Select APS (Alternative Protein Source) LB Media” was prepared bydissolving 20 g of Select APS LB broth base powder in 1 L of purifiedwater with a pH that ranged from about 6.6 to about 7.1. LB agar plateswere prepared by adding 7.5 g of Agar, U.S.P. into 500 mL of Select APSLB media. Both APS LB media and agar were autoclaved for 121° C. to 123°C. for ≧45 min on liquid cycle. After cooling to 40-60° C., Kanamycinantibiotic was added to the media and agar plates at a concentration of50 μg/mL.

B. Host Cell Transformation

Plasmid GDF5-T5 or pGDF5-Trc DNA (SEQ ID NOS:2 and 3, respectively) anda competent cell E. coli host cell, either from strain DH10β, STBL2,HMS174 or recA, were each separately mixed together in a tube andincubated on an ice bath for at least about 30 minutes, heat shocked for45 seconds at 42° C.±2° C.) and immediately placed on ice for 2-5 min.About 450 μl of SOC media (Bacto tryptone 20 g/L; Bacto yeast extract, 5g/L; NaCl, 0.5 g/L; MgCl₂.6H₂O 2.03 g/L; glucose 3.6 g/L) was then addedinto each bacteria-plasmid DNA mixture and placed onto 37° C. (±1° C.)shaker incubator and shaked at about 225 to about 275 rpm for 60 minutes(±5 minutes). To prevent lowering of a dissolved oxygen concentration,the shaker incubator was sped up to keep the dissolved oxygenconcentration at 50% of air saturation. After 1 hour, an aliquot of thetransformed cells were aseptically plated into APS LB/Kn agar plates andincubated overnight at 37° C. (±1° C.) for about 14 to about 24 hours.The cultivation was proceeded by adding 50% glucose solution at a levelof 0.2% to obtain a high cell density, with an indication of abruptincrease of the dissolved oxygen concentration. The final pH of thegrowth medium was at about a pH of 7.

Table 4 lists the different transformed groups with their correspondinghost cell strain and vector constructs:

TABLE 4 Transformed Groups Host Cell Line Vector Construct A DH10β(Clones 1-5) pGDF5-T5 B DH10β (Clones 1-5) pGDF5-Trc C STBL2 (Clones1-5) pGDF5-T5 D STBL2 (Clones 1-5) pGDF5-Trc E HMS174 (Clones 1-5)pGDF5-T5 F HMS174 (Clones 1-5) pGDF5-Trc G recA (Clones 1-5) pGDF5-T5 HrecA (Clones 1-5) pGDF5-Trc

pGDF5-T5 and pGDF5-Trc constructs were each successfully transformedinto each four host cell lines. Clonal selection and expressionscreening were conducted by first performing a small scale fermentationwith IPTG induction (1 mM final concentration). For STBL2, clonalselection was performed at 30° C. The presence of pGDF5-T5 or pGDF5-TrcDNA (SEQ ID NOS:2 and 3, respectively) was each confirmed by restrictionenzyme analysis with NdeI. GDF5 protein analysis was assessed throughSDS-PAGE and Western blotting

To monitor the transformation process, a positive (+) control vectorconstruct, pJExpress, was used. A clone that expresses the pJExpressconstruct showed an over-expression of a fluorescent protein of about 30kDa protein after IPTG induction (data not shown).

Expression Screening of pGDF5-T5 and pGDF5-Trc Constructs

A. DNA-Restriction Enzyme Analysis:

Five bacterial colonies (Clones 1-5) from each transformed groups,Groups A-H as listed in Table 4, were initially picked for overnightgrowth in APS LB media containing 50 μg/ml of Kanamycin (APS LB/Kan).The next day, two (2 ml) of APS LB/Kan bacterial culture from eachcolony of each transformed groups, were grown at 37° C. (±1° C.)overnight. Plasmid DNAs were extracted and purified using a QIAprep®Spin Miniprep Kit (Qiagen, Inc, Valencia, Calif.). The purified DNAswere linearized with NdeI restriction enzyme and run through a 0.8%agarose gel to verify for the presence of the pGDF5-T5 and pGDF5-Trcconstructs. Both linearized pGDF5-T5 and pGDF5-Trc DNAs have an expectedsize of 4299 bp and 4403 bp, respectively. As shown in FIGS. 2A and 2B,successful transformation of pGDF5-T5 and pGDF5-Trc DNA into DH10β andSTBL2 (grown at 30° C.) host cells clones 1-5, respectively, wasconfirmed. Transformation of pGDF5-T5 and pGDF5-Trc (Clones 1-5,respectively) on HMS174 and recA host cell strains was also successful,the results of which are not presented herein.

B. Preparation of E. coli Inclusion Bodies (IB)

Positive transformants from each Group (A-H) as listed in Table 4 werecultured according to the above-mentioned method. Cells from the culturebroth of each transformant were harvested and resuspended in TE buffer(25 mM Tris and 10 mM EDTA, pH 7.3). To collect inclusion bodies thatcontain the highly purified concentrated rhGDF-5 protein, cells werebroken up by means of a homogenizer and spun down to collect the pellet(or precipitate) that contains the inclusion bodies. The inclusionbodies were washed with wash buffer and centrifuged for a period of timeat 4° C. The collected pellet was solubilized by sonicating insolubilization buffer. After solubilization, the solution containing therhGDF-5 protein was centrifuged for a period of time at 4° C.

To obtain high purity with the highest maximum yield rhGDF-5 protein yetlow oxidation and minimal related impurities, the resultant supernatantwas subjected to a weak cation exchanger resin, Toyopearl™ CM-650 fromTosoh Bioscience LLC, King of Prussia, Pa. Briefly, the CM-650 columnwas first equilibrated with buffer before the resultant supernatant wasapplied to the CM-650 column. The CM-650 column was washed beforeeluting with the same buffer modified with a salt to elute the proteinsoff the column. In various embodiments, Toyopearl™ CM-650 may bereplaced by Amberlite® IRC86, CM agarose-based material and/or Dowex® asthe weak cation exchanger resin.

In various embodiments, various washing buffers can be used during thepurification process such as, for example, phosphate buffer saline(PBS). In some embodiments, GDF-5 protein purity is approximately 95%,96%, 97%, 98%, 99% and/or 100%.

C: SDS-PAGE and Western Blotting

To screen for bacterial clones capable of expressing rhGDF-5 protein,five single colonies from each group as listed in Table 1, were furtherinoculated in 4 ml of APS LB agar plates containing 50 μg/ml ofKanamycin (APS LB/Kan) and grown at 37° C. When the bacterial culturegrew to OD₆₀₀ from about 0.4 to about 0.6, they were induced with orwithout IPTG (1 mM final concentration; ±IPTG). Non-induced and Inducedcultures were each harvested and treated with Novagen™ BugBuster ProteinExtraction Reagent. Supernatant (soluble proteins) and pellet (insolubleproteins) from each culture, either IPTG-induced or uninduced, were thenanalyzed by SDS-PAGE.

For SDS PAGE, a total of 20 μL sample (13 μl of sample, 2 μl of reducingagent and 5 μl of sample loading buffer) was prepared for gel loading.The 20 μL-samples were boiled at 95° C. for 5 minutes and loaded eitheras 5 μl or 20 μl for larger volume loading. All positive clones showedthe presence of the expected 13.5 kDa rhGDF-5 protein.

rhGDF-5 expression was observed in the pellets of clones from all of thehost cell strains tested, regardless of which vector constructs was usedin the transformation. Overexpression of rhGDF-5 protein wasparticularly observed in clones from HMS174 and RecA strains and wasbetter than those of DH10β and STBL2 ((data not shown). In addition, ahigher rhGDF-5 over-expression was observed with longer IPTG inductiontime (results not included). The size-wise comparison of theover-expression band with the reference protein, rhGDF5 was furtherconfirmed by Western blotting analysis using an anti-human GDF-5antibody.

From the Western blot results obtained, GDF-5 protein was found in thebacterial pellet fraction (see FIG. 3B) and not in the supernatantfraction (see FIG. 3A). Also, the expression level using the GDF5-TRCconstruct was better than that of pGDF5-T5 construct. Among the 4different host cell lines expressing pGDF5-TRC, the HMS174 cell strainprovided the optimally-expression levels of the GDF-5 protein.

Characterization of the Over-Expressing GDF-5 Clones

About 400 μl±2% of thawed pGDF5-Trc- or pGDF5-T5-transformed clones wereaseptically and inoculated into two separate flasks of pGDF5-T5 andpGDF5-Trc-transformed RecA or HMS174 (Flask 1 for growth monitoring andFlask 2 for storage processing) in the growth medium. Each of theinoculated flasks were shaked at 220 to 250 rpm at a temperature ofabout 37° C.±1° C. until an optical density (OD₆₀₀) of about 1 to about3 was reached. After 8 hours of fermentation (EFT8; Elapsed FermentationTime), hourly sampling from Flask 1 was taken for optical densitymeasurements. Sampling may occur prior to EFT 8 and when OD₆₀₀ of Flask1 reached between 0.8 and 1.0, the OD₆₀₀ of Flask 2 was also measured.

Of the forty clones (5 clones per each transformed group) examined, twoclones, Clones #1 and 4 derived from pGDF5-TRC-transformed HMS174, wereselected and evaluated for further studies, e.g., ability to express andproduce GDF-5 and growth profile and rate (see FIGS. 4A and 4B,respectively).

The growth profile study was done to understand how these cells grow inthe lag, exponential and stationary phases. The data obtained may thenallow for profiling growth rates, population doubling time (expressed asp), carbon consumption and waste production.

Growth rate constant, according to the embodiment of the presentdisclosure, can be defined as the number of generations that occur perunit time (expressed as μ or mu), where (1) μ=(ln N2−ln N1)/(t2−t1)where N2 and N1=cells ml-1 at time t2 and t1 (in h); and (2) convert (1)to log: μ=(log N2−log N1) (2.303)/t2 and t1).

As depicted in FIG. 4A, pGDF5-Trc-transformed HMS174 Clones 1 and 4 bothoverexpress the rhGDF-5 protein. However, the growth profile of Clone 4was better than that of Clone 1 (with Clone 4 having a higher mu or μvalue for growth rate), despite the similarity in their OD₆₀₀ valuesstarting from EFT0 to EFT18.5-19.0 (see FIG. 4B). Growthcharacterization and protein production were further conducted bygrowing the transformants using two fermentation methods: (1) ultrayield shake flask (UY SF) and (2) 5 L Applikon Fermentor (Ferm).

The ultra yield shake flask (UY SF) fermentation method, as discussedabove, may be used to easily make the recombinant protein materialwithout the use of a fermentor. This technology is disposable, and isproduct dedicated. It stimulates a fermentation environment but does notrequire the infrastructure and laborious setup that actual bioreactorfermentations need. It has a similar set up like a disposable shakeflask and allows for the manufacturing of the recombinant proteinmaterial that is closely representative of what can be made in afermentor to support downstream and analytical development of therecombinant protein

The 5 L fermentation method, on the other hand, is a non-GMP batchinduction fermentation method for production of the recombinant GDF-5protein. It involves the inoculation of 400 mL of seed media with 1000μl seed ampoule of pGDF5-Trc-transformed Clone 1 or 4 and shaking themedia at 250 rpm, for 8 hrs at 37° C. This was followed by inoculatingabout 200 mL of seed culture into the 5-L fermenter with the followingfermentation parameters:

Fermentation Parameters: Temperature 37° C., Stirring: 330-1322 rpm,Airflow: 4 L/min, pH control: 6.8+/−0.2 with ammonium hydroxide and 50%phosphoric acid, dissolved oxygen: 30%, and anti-foam 204 as needed.

When the OD₆₀₀ reaches at about 0.6-0.8, 1 mM IPTG was added forinduction. After 14-18 hours of post-induction, the bacterial culturewas harvested.

The goal for exploring these methods is to develop a recombinant proteinproduction that is not laborious and simple for analytical andpurification approach. These two methods vary in terms of the followingas illustrated in Table 5 and in the results obtained as shown in FIG.5A and FIG. 5B.

TABLE 5 UY Shake Flask 5 L Applicon Fermentor (FIG. 6A) (FIG. 6A) pHControl APS Super Broth with APS Super Broth with 100 mM MOPs H3PO4 andNH4OH pH Shift pH shift to more acidic may pH maintained at causeprotein induction 7.0 ± 0.2 Growth lower growth rate growth rate wasabove 1 Rate at induction at induction

A better GDF5 protein expression was observed when UY SF method wasemployed (see SDS-PAGE as shown in FIG. 6A), but the overall GDF5expression may appear equivalent between Clones 1 and 4 (see westernblot as shown in FIG. 6B, lanes 6 and 10). In addition, the pelletfraction from Clone 1 UY SF (sample PF030311A, lane 10) appeared to havea more dense GDF5-band (see FIG. 6B). Supernatant (soluble proteins) andpellet (insoluble proteins) samples from pGDF5-Trc-transformed Clones 1and 4 were analyzed using SDS-PAGE and have the following designations:

TABLE 6 Sample Designated Sample Source Description pGDF5-Trc Clone #Supernatant SF020311A Clone 1 UY SF Pellet PF020311A Clone 1 UY SFSupernatant SF020311B Clone 4 UY SF Pellet PF020311B Clone 4 UY SFSupernatant SF020311C Clone 1 5L Ferm Pellet PF020311C Clone 1 5L FermSupernatant SF020311D Clone 4 5L Ferm Pellet PF020311D Clone 4 5L FermMaximizing pGDF5-Trc DNA Yield and Optimal Recombinant GDF-5 ProteinProduction

High plasmid DNA yield and recombinant protein production, while stillcost-effective, are important for the manufacturing of rhGDF-5biologics. Therefore, to attain the maximum pGDF5-Trc DNA yield andrhGDF-5 protein production, efforts were made to obtain a high celldensity fermentation for DNA production. Specifically, the type ofproduction (batch, fed-batch or continuous fermentation), type of mediaand components and growth control strategies were considered. To achievethese goals, the inventors added several components or ingredients to ahigh cell density media (formulation as described in FIG. 7) todetermine their effects of cell growth, GDF5-Trc DNA plasmid yield andrhGDF-5 protein expression without compromising GDF5-Trc plasmid qualityand GDF-5 protein expression. A two level fractional factorial designedof experiment (DoE) was performed in Thomson 24-microwell plates usingeither a defined (minimal) or semi-defined (complex) media. Statisticalsoftware was employed to evaluate these various formulations forfermentation media.

A. Defined Media DoE (7-Factor DoE Design Space)

The defined media additionally includes ingredients such as sodiummolybdate (ranges from about 5 to about 10 mg/L), magnesium sulfateheptahydrate (ranges from about 2 to about 6 mM), sodium chloride(ranges from 0 to about 4 g/L), EDTA (ranges from 0 to about 400 mg/L),MOPS (ranges from 0 to about 100 mM), amino acid supplement includingL-methionine (ranges from 0 to about 10 ml/L) and vitamin supplement(folic acid, pyrodoxine, and biotin; ranges from 0 to about 10 ml/L).Center points were added to detect for curvature and residual testingand lack-of-fit testing were included in the studies. The transformedbacteria were grown in Thomson 24-well microplate at 37° C., 250 rpm,induced after 4 hours of elapsed fermentation time (EFT4) with 1 mM IPTGand harvested after incubating for an additional 14 hours. Chemicallysis was performed on harvested samples. Bacterial pellet samples wererun on SDS-PAGE. Gels were analyzed using ImageJ densitometry software(see FIGS. 8A-B).

B. Semi-Defined Media DoE

The semi-defined (complex) media included, in addition to what was inthe defined media, yeast extract and tryptone (animal-derived). Bothyeast extract and tryptone range from 0 to about 0.4% w/v. Also includedwere sodium molybdate, magnesium sulfate, sodium chloride, EDTA, MOPS(3-[N-morpholino] propane-sulfonic acid), amino acids (includingL-methionine), and vitamins (folic acid, pyrodoxine, and biotin). Thecenter points were added to detect for curvature and residual testingwere included. The transformed bacteria were grown in Thomson 24-wellmicrotitreplate in high-throughput minibioreactor system at 37° C., 1000rpm. During this time, biomass, pH and pO2 were measured at 15 minuteinterval. The bacteria culture was induced after 4 hours of elapsedfermentation time (EFT4) with 1 mM IPTG and harvested after incubatingfor an additional 14 hours. Chemical lysis was performed on harvestsamples and bacterial pellet samples were run on SDS-PAGE and analyzedusing ImageJ densitometry software (see FIGS. 9A-D).

Based on the results obtained from the two-level fractional factorialdesign, three other high cell density (HCD) growth media were tested andfurther developed. They differ from each other with respect to theiroptimized response to either rhGDF5 expression (protein production) orbiomass yield (growth rate) as follows: Media 1 (defined media improvedrhGDF-5 expression) included sodium molybdate, magnesium sulfate andsodium chloride. Expression of rhGDF-5 was increased by increasingsodium molybdate, magnesium sulfate and sodium chloride in defined media(see Tables 6-7 and FIGS. 8A-B). Media 2 (semi-defined media improvedrhGDF-5 expression) included magnesium sulfate and yeast extract.Expression of rhGDF-5 was increased in the presence of yeast extract andmagnesium sulfate but decreased when sodium molybdate was present (seeTables 7-8 and FIGS. 9A-B). Media 3 (semi-defined media improved biomassyield) included yeast extract and tryptone while being negativelyaffected by sodium chloride and MOPS (see Tables 7-8 and FIGS. 9C-D).

As demonstrated in Table 7 and 8, Media 1 and 2 both enhanced theexpression of rhGDF-5 while Media 3 improved or optimized biomass yield.

TABLE 7 Optimized Harvest rhGDF-5 Band Amount Media Media Type ResponseOD₆₀₀ (μg/mL) 1 Defined rhGDF-5 7.58 0.133 expression 2 Semi-DefinedrhGDF-5 8.3 1.03 expression 3 Semi-Defined Biomass 9.06 0.040 4 SuperBroth NA 15.1 0.105 (Complex)

It was also noted that scale-up to 1 L volumes precipitated Media 2while reduction in phosphates allowed the expression of rhGDF-5 andprevented precipitation of magnesium phosphate. Furthermore,substituting tryptone with peptone, a non-animal derived alternative,increased rhGDF-5 expression. Data not included herein.

Similar findings were obtained with a smaller scale fermentation(24-well plate) and the 2.5 L Ultra Yield Flask scale up fermentationmethods with respect to the effect of Media 1 and 3 on GDF5 on biomassyield (see Table 8). Their effect on GDF-5 protein expression was notthe same. Enhanced GDF5 protein expression was demonstrated whencultures were grown on the 24-well plate with Media 2 but not on Media 1and Media 3. See data from Table 8. A problem was encountered with Media2 during the 2.L-Ultra Yield Flask scale-up fermentation run. Thesolution precipitated during overnight storage which may have been dueto magnesium sulfate reacting with phosphate-buffering system to forminsoluble magnesium phosphates.

TABLE 8 Media Alternatives 2.5 L UY Confirm. Run 24 Well Plates rhGDF-5rhGDF-5 rhGDF5 rhGDF-5 rhGDF 5 rhGDF-5 band amount per band amount perband amount per Media Optimized Harvest amount ml ferm Harvest amount mlferm Harvest amount ml ferm Media Type Response ID OD600 (μg) (μg/ml)OD600 (μg) (μg/ml) OD600 (μg) (μg/ml) 1 Defined rhGDF-5 N/A 8.64 0.667814.99 7.25 2.596 24.15 7.58 0.1402 1.023 expression 2 Semi rhGDF-5 2a9.82 0.1913 6.278 7.79 0.4379 4.378 8.3 0.9934 7.936 defined expression2b 9.82 0.7756 16.37 3 Semi Biomass 3  11.96 0.1406 10.11 12.2 3.34352.34 9.06 0.0351 0.3060 defined 3a 10.12 0 0 3b 10.42 0 0 3c 11.421.172 17.17   5B Super N/A N/A 22.72 1.609 46.92 19.06 2.181 53.34 15.10.1668 2.425 Broth (Complex)Effects of pH and Oxygen on the GDF-5 Protein Production and Expression

To study the effects of pH and oxygen on GDF-5 protein production andexpression, pGDF5-Trc-transformed host cells (HMS174 strain, CloneF031512 were grown under the following growth conditions: (i) pH 6.5(condition A); ii) pH 7.1 (condition B); and iii) pH 6.8 at low oxygen(condition C) and induced with IPTG. Samples designated hereinafter asF031512A, F031512AB, and F031512A C from each of the three conditionswere normalized to 4.0 at OD₆₀₀ by dilution into BugBuster® PlusLysonase™ (Novagen®) before separately running them under reducing andnon-reducing SDS-PAGE conditions. Each of the sample pellets (EFT22,EFT24, EFT26 and EFT28 were resuspended in equal volume of water andadded to 4× reducing sample buffer. All 3 SDS-PAGE gels (see FIGS.11A-C), i.e., Samples F031512A, B, and C loaded on Gels A, B, and C,respectively, were stained and de-stained in the same gel tray.Intensity and banding pattern of the reference standards varied greatlyfrom gel to gel. Densitometry data was generated comparatively. Allcalculations were based on standards value from the gel that produced asingle band standard (Gel C). Though the overall yield estimationsaccuracy maybe questionable but their relative comparisons should bevalid since the molecular weight ladder showed consistent band intensityat molecular weights similar to the protein of interest.

As shown in FIG. 11E, the F031512A samples resulted in a higher ratio ofthe two major expressed protein bands (40 kDa:14 kDa) than SamplesF031512B and F031512C. Low oxygen (condition C) resulted in the lowestratio of 40 kDa:14 kDa (FIG. 11E). Among the three conditions tested,cells that grew under growth media of pH 7.1 (condition B) had thehighest overall GDF5 protein expression (FIG. 11B) and growth rate (seeFIG. 11D) than cells that grew under condition A (growth media of pH6.5; see FIG. 11A) and condition C (low oxygen condition; see FIG. 11C).Extreme reduction in dissolved oxygen leading to anaerobic conditionsmay not improve protein expression but can influence the ratio of 40kDa:14 kDa (see FIGS. 11C and 11E).

It will be apparent to those skilled in the art that variousmodifications and variations can be made to various embodimentsdescribed herein without departing from the spirit or scope of theteachings herein. Thus, it is intended that various embodiments coverother modifications and variations of various embodiments within thescope of the present teachings.

What is claimed is:
 1. A host cell line, the host cell line comprisingan inclusion body comprising a GDF-5 protein, wherein the host cell linefurther comprises an expression vector comprising a polynucleotideencoding the GDF-5 protein under the control of a T5 promoter, and theGDF-5 protein is a recombinant human GDF-5 (rhGDF-5) protein comprisingthe amino acid sequence of SEQ ID NO:
 4. 2. A host cell line of claim 1,wherein the expression vector further comprises a kanamycin resistance(Kan^(r)) gene and a pUC origin of replication.
 3. A host cell line ofclaim 1, wherein the T5 promoter is inducible byisopropylthio-β-galactoside (IPTG).
 4. A host cell line of claim 1,wherein the vector is a plasmid vector that is pGDF5-T5.
 5. A host cellline of claim 4, wherein the pGDF5-T5 comprises the polynucleotidesequence of SEQ ID NO:
 2. 6. A host cell line of claim 1, wherein: theT5 promoter is inducible by isopropylthio-β-galactoside (IPTG); and thevector is pGDF5-T5.
 7. A host cell line of claim 6, wherein the pGDF5-T5comprises the polynucleotide sequence of SEQ ID NO:
 2. 8. A host cellline of claim 1, wherein the cell line comprises a prokaryotic cell andwherein the inclusion body is from an Escherichia coli strain comprisingof DH10β, STBL2, HMS174 and RecA.
 9. A host cell line of claim 1,wherein the cell line is in a medium comprising from about 5 mg/L toabout 10 mg/L sodium molybdate.
 10. A host cell line of claim 1, whereinthe cell line is in a medium comprising from about 2 mM to about 6 mMmagnesium sulfate heptahydrate.
 11. A host cell line of claim 1, whereinthe cell line is in a medium comprising sodium chloride.
 12. A host cellline of claim 1, wherein the cell line is in a medium comprising aminoacid supplement including L-methionine.
 13. A host cell line of claim 1,wherein the cell line is in a medium comprising a vitamin supplementthat includes folic acid, pyridoxine, and biotin.
 14. A prokaryotic hostcell line, the host cell line comprising an inclusion body comprising aGDF-5 protein, wherein the host cell line further comprises anexpression vector comprising a polynucleotide encoding for the GDF-5protein under the control of a T5 promoter, and the GDF-5 protein is arecombinant human GDF-5 (rhGDF-5) protein comprising the amino acidsequence of SEQ ID NO:
 4. 15. A host cell line of claim 14, wherein: theexpression vector further comprises a kanamycin resistance (Kan^(r))gene and a pUC origin of replication.
 16. A host cell line, the hostcell line comprising an inclusion body comprising a GDF-5 protein, thehost cell line further comprising an expression vector comprising apolynucleotide encoding for the GDF-5 protein under the control of a T5promoter that is inducible by isopropylthio-β-galactoside (IPTG), theexpression vector further comprising a kanamycin resistance (Kan^(r))gene and a pUC origin of replication, wherein the GDF-5 protein is arecombinant human GDF-5 (rhGDF-5) protein having the amino acid sequenceof SEQ ID NO: 4, and wherein the vector is pGDF5-T5 comprising thepolynucleotide sequence of SEQ ID NO: 2, and the host cell line is froman Escherichia coli strain comprising DH10β, STBL2, HMS174 and RecA.